PRINCIPLES OF TOXICOLOGY

256 MUTAGENESIS AND GENETIC TOXICOLOGY
Tests for Primary DNA Damage
A historical test thought to monitor primary DNA damage in mammalian germ cells in vivo involves
the monitoring of sister chromatid exchange. The observation of sister chromatid exchanges through
differential staining involves exposing the cells to bromodeoxyuridine for two rounds of replication,
so that the chromosomes consist of one chromatid substituted on both arms with 5-bromodeoxyuridine
and the other substituted only on a single arm. Differential staining between sister chromatids is due
to the differences in bromodeoxyuridine incorporation in the sister chromatids.
Unscheduled DNA repair has been induced by chemical mutagens in mammalian male germ cells
from the spermatogonial to midspermatid stages of development. The test is based on the fact that cells
not undergoing replication (scheduled DNA synthesis) should not exhibit significant DNA synthesis.
Thus, incorporation of radiolabeled tracer molecules into the DNA of these cells should be minimal.
However, if a chemical mutagen damages the DNA, the DNA repair system may be activated, causing
unscheduled DNA synthesis (UDS). If such is the case, radiolabeled tracers will be incorporated into
the DNA; these can be monitored by autoradiography or by direct measurement of radioactivity in the
repaired DNA. Male germ cells lose DNA repair capability when they have advanced to the late
spermatid and mature spermatozoa stages; unscheduled DNA synthesis cannot then be induced by
chemical mutagens. The genotoxic agents methyl methanesulfonate, ethyl methanesulfonate, cyclophosphamide,
and Mitomen have been shown to induce unscheduled DNA repair in vivo in male mouse
germ cells. Similar procedures are available to evaluate UDS in some types of somatic cells as well.
Transgenic Mouse Assays
In the late 1980s and early 1990s the development of a new genotoxicity assay system was reported
by Gossen et al. (1989), Kohler et al. (1991), and colleagues. Briefly, the test system involves mutagen
dosing to a specific mouse strain (C57BL6) that has been infected with a viral “shuttle vector,”
isolation of the mouse DNA, recovery of the phage segment (lacI or LacZ), and infection of an E. coli
strain with the recovered phage. The phage will form plaques on a lawn of E. coli. The plaques are
colorless if no mutation has occurred or blue if a mutation has occurred. The assay may be performed
to gather information on mutations in somatic cells or in germ cells. The lacZ assay also is known as
the “Muta-Mouse” assay, while the lacI assay also is known as the “Big Blue” assay.
Advantages of the assay include its in vivo treatment regime, the fact that it can be conducted in a
few days from the isolation of DNA through plaque formation to mutation scoring. However, it may
be difficult to use extremely high dosages (e.g., approaching lethal doses), since the mice must survive
for 1–2 weeks in order to fix the mutation in the affected tissues. The performance of the transgenic
mouse assays that have been conducted on 26 substances was evaluated by Morrison and Ashby (1994),
including a review of the results of the tests that had been performed in the lacZ case (14 reports) and
in the lacI case (16 reports). These authors concluded that the variability of data reporting formats and
the rapid developments and modifications in the assay protocols make it difficult to perform direct
comparisons among tests or between this assay type and the results of other historically available
methods. Nevertheless, the initial results are generally promising, and there are no examples of internal
disagreement between responses for the same chemical in the same tissue.
In Vitro Testing
Test systems have been developed that use mammalian cells in culture (in vitro) to detect chemical
mutagens. Disadvantages in comparison with in vivo mammalian tests are that in vitro tests lack
organ–system interaction, require a route for administration of the agent that cannot be varied, and
lack the normal distributional and metabolic factors present in the whole animal. The obvious
advantages are that costs are decreased and that experiments are more easily replicated, which
facilitates verification of results. Cases where human cells have been cultured successfully (e.g.,
lymphocytes) provide the only viable in vitro experiments on the human organism. Several endpoints